This invention relates to a designing of PCR based detection method for chlamydia trachomatis. 
Culture method: Chlamydiae were first detected by light microscopy in conjuctival scrapings from orangutangs inoculated with material from trachoma patients in 1907. The diagnostic sensitivity and specificity of light microscopy, however, are not satisfactory. Because Chlamydia depends on ATP and other nutritional factors from a host cell it can reproduce only in other cell. The inclusion body, containing thousands of C. trachomatis can be visualized by staining with fluorescein-conjugated antibody directed against one of the organism's surface antigens. Because the inclusion body is highly characteristic, cell culture is considered to have a specificity of 100%.
Sensitivity of this method is as low as 50% as organisms may lose infectivity during transportation and storage, which will reduce the likelihood of propagation. In addition, the surface area of the cell culture layer and/or the amount of sample material added to the cell culture influence the sensitivity. Cell culture, however, is time-consuming, laborious and expensive and can therefore be provided by only a few central laboratories.
Antigen detection: Antigen detection methods comprise Enzyme-inked immunosorbent assays (ELISA) and Direct immunofluorescence assays (DFA). The currently commercially available ELISA all use the LPS as antigen. The LPS part of Chlamydia binds to immobilized anti-LPS antibodies and the ELISA tests are therefore genus specific and detect all Chlamydia species. A secondary antibody that is bound to the Chlamydia is linked to an enzyme, which generates a colour change, measured as optical density, on addition of subtrate. In DFA, fluorescein-conjugated antibodies directed against either the LPS or the MOMP component react with the Chlamydia surface. The fluorescein can subsequently be visualized by fluorescence microscopy.
The diagnostic efficacy of these methods is not high enough to warrant clinical use unless the need for a fast result overweighs the lower diagnostic accuracy. Also, the ELISA tests may reveal positive results in the presence of other organisms such as E. coli and Bacteroides sp, and Staphylococcus aureus may be captured instead of Chlamydia due to binding to the Fc region of the antibodies, thereby causing false-positive reactions. DFA requires skilled personnel in order to differentiate C. trachomatis organisms from non-specific fluorescent particles.
DNA/RNA detection: DNA/RNA detection assays can be divided into probe assays and amplification assays. In probe assays a synthesized single stranded oligonucleotide hybridizes to a part of C. trachomatis DNA or RNA. The most widely used probe technique is the Gen-Probe assay, in which a probe reacts with ribosomal RNA (rRNA) of C. trachomatis, which is present in hundreds of copies in each organism.
The diagnostic performance of non-amplified probe technique is not substantially different from that of the best ELISA.
Nucleic acid amplification tests (NAATs): In nucleic acid amplification Tests specific probes hybridize to C. trachomatis DNA or RNA and the DNA/RNA flanked by the primers (target DNA) is exponentially copied. Target gene—The Plasmid: The plasmid is unique for C. trachomatis, is well conserved within the species, and is present in approximately 10 copies in each C. trachomatis organism. Using the plasmid as target DNA should therefore theoretically lower the detection limit by a factor of 10 compared with a single chromosomal gene, for example MOMP gene.
The 16S-rRNA gene: By using the 16S-rRNA gene, which is present in all bacteria and is the most conserved gene known, all Chlamydia species can be detected by just one primer set. This is done by constructing the primers to anneal at the genus-specific regions of the 16S-rRNA gene. The genus specific regions flank variable-regions that are specific for each species. The amplified products comprise the variable region flanked by the two genus specific regions. The species can be determined by specific probe or RFLP, or by DNA sequencing.
Target gene for NAATs Target gene for NAATs The Plasmid: Some studies give evidence or suggest that the plasmid-free variants are present in clinical samples, and although it may seem that plasmid is involved in DNA replication, it has been possible to culture a plasmid-free variant. Thus, the infections caused by plasmid-free variants will be undetected if the plasmid is used as target gene.
The 16S-rRNA gene: Due to high homology of the 16S RNA gene with other organisms, optimal reaction conditions are crucial in order to avoid annealing of primers to 16S-rRNA gene of the other organisms that are present in all non-sterile clinical samples.